Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a pathogen of global importance; 9 million new cases of TB are reported annually, with 1-2 million deaths. There are half a million cases of drug resistant TB cases each year and the problem of antibiotic resistance is rising. TB treatment is a lengthy process (6-24 months) giving rise to an urgent need for newer, more effective antibiotics. In order to develop new drugs, we need to understand which metabolic processes are the most important for bacterial survival and pathogenesis. Chemical entities can be powerful probes for the identification of key cellular processes underpinning survival. However, most recent work has focused on genetic approaches to identifying essential genes and there has been little progress in identifying the pathways that effective anti-bacterial compounds target. Recently, a number of novel compounds with unknown targets have been identified with cidal or static activity against drug-resistant pathogens including M. tuberculosis. We propose to apply a combination of techniques in conjunction with several compound classes in order to identify vulnerable pathways, which can be thoroughly characterized using compounds as chemical probes. We will use three main approaches to characterize anti-tubercular agents. (1) We will isolate compound-resistant strains (spontaneous mutants or over-expressing recombinant strains) to identify the protein targets and mode of resistance. (2) We will use three methods (affinity chromatography, photoaffinity labeling, and yeast three hybrid system) to identify protein targets which bind to the compounds. (3) We will look at the effect of compound exposure on cell metabolites and the induction of reactive oxygen species. Together these methods will enable us to identify the pathways and specific proteins that are targeted by each compound; such targets will form the basis for future drug discovery and development.